Animals with insufficient expression of uridine phosphorylase

ABSTRACT

Non-human animals deficient in a function of a uridine phosphorylase gene on a chromosome, and their offspring. These non-human animals and their offspring make it possible to elucidate pathologic functions and activities of nucleic acid dysbolism and the like and also to predict utility of pyridine nucleoside antimetabolites in human, and are useful as experimental animals.

TECHNICAL FIELD

This invention relates to non-human animals deficient in the expressionof uridine phosphorylase, and also to their offspring.

BACKGROUND ART

Pyrimidine nucleoside phosphorylases are enzymes, which take part in thebiosynthesis and degradation of pyrimidine in its metabolism and play animportant role of regulating the in vitro nucleoside pool throughdegradation and synthesis of pyrimidine bases in the salvage pathway.Further, pyrimidine nucleoside phosphorylases in mammals are known toinclude uridine phosphorylase and thymidine phosphorylase, both of whichtake part in the biosynthesis and degradation in its metabolism.Concerning such pyrimidine nucleoside phosphorylase genes, cDNAs havebeen isolated from mice and human, and elucidation of their differencesin expression is now under way on the gene level [Uchida et al., J.Biol. Chem., 270, 12191-19196 (1995); Uchida et al., Biochem. Biophys.Res. Commun., 216, 265-272 (1995)].

On the other hand, nucleoside antimetabolites which are playing animportant role in the field of cancer chemotherapy in recent years areknown to be inactivated by pyrimidine nucleoside phosphorylase due totheir chemical structures. For the development of drugs excellent inpharmacological effects and low in side effects, it is thereforeconsidered to be necessary to accurately grasp the mechanism ofmetabolism of these substances by pyrimidine nucleoside phosphorylase.

Uridine phosphorylase and thymidine phosphorylase are, however, observedto have substantial differences in their distribution depending onspecies. In human, thymidine phosphorylase are expressed in both normaltissues and tumor tissues, whereas in rodents such as mice and rats,uridine phosphorylase is primarily expressed centering around digestivetracts and thymidine phosphorylase is expressed only in some tissuessuch as livers. This has led to a problem in that the utility and thelike of nucleoside anticancer agents in human cannot be preciselypredicted from data on rodents.

On the other hand, thymidine phosphorylase is known to function as anangiogenic factor. It has recently been identified as a causative geneof MNGIE, a mitochondrial disease.

Accordingly, uridine phosphorylase expression deficient animals, ifavailable in a phyletic lineage, will be useful as experimental animalsfor the study of physiological functions of the protein, for theelucidation of its related diseases, and also for the development andresearch of pyridine nucleoside antimetabolites (anticancer agents).

An object of the present invention is to provide a uridine phosphorylaseexpression deficient non-human animal or its offspring, which can beused as an experimental animal.

DISCLOSURE OF THE INVENTION

With the foregoing current circumstances in view, the present inventorshave proceeded with research in various ways on mutation of a uridinephosphorylase gene, designing of a targeting vector, and so on. As aresult, the present inventors have succeeded in creating a transformedanimal having a uridine phosphorylase gene which practically does notfunction, and its offspring, leading to the completion of the presentinvention.

Specifically, the present invention provides a non-human animaldeficient in a function of a uridine phosphorylase gene on a chromosome,or an offspring thereof.

As a result of developments in genetic engineering in recent years, ithas become possible to artificially manipulate various genes and tocreate various transformed animals artificially added with foreigngenetic characteristics or controlled in the expression of geneticcharacters which the organisms inherently possess [Nature, 300, 611-615(Dec. 16, 1982); Proc. Natl. Acad. Sci. USA, 87, 7688-7692 (October1990), etc.]. However, animals which are deficient in the function of auridine phosphorylase gene on a chromosome and are genetically stableare not known to date.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a mouse uridine phosphorylasegene and its targeting disruption. In the representation, neo representsa neomycin-resistant gene, DT-A represents the gene of fragment A ofdiphtheria toxin, LacZ represents a β-galactosidase gene, thick solidlines represent exons, and fine solid lines represent introns.

FIG. 2 is a construction scheme of a targeting vector for the mouseuridine phosphorylase gene. In the scheme, neo represents theneomycin-resistant gene, DT-A represents the gene of fragment A ofdiphtheria toxin, LacZ represents the β-galactosidase gene, thick solidlines represent exons, and fine solid lines represent introns.

FIG. 3 illustrates electrophoresis patterns showing the results ofgenomic Southern hybridization which was conducted for the confirmationof homologous recombinant ES cells.

FIG. 4 illustrates electrophoresis patterns showing the results ofgenomic Southern hybridization which was conducted for the confirmationof a homologous recombinant mouse. In the electrophoresis patterns, +/+indicates a normal mouse, +/− indicates a heterozygous deficiency mouse,and −/− indicates a homozygous deficiency mouse.

FIG. 5 illustrates electrophoresis patterns showing expression levels ofuridine phosphorylase by normal mice and uridine phosphorylaseexpression deficient mice. In the electrophoresis patterns, +/+indicates a normal mouse, and −/− indicates a homozygous deficiencymouse.

BEST MODES FOR CARRYING OUT THE INVENTION

The expression “deficient in a function of a uridine phosphorylase geneon a chromosome” as used herein means that a uridine phosphorylase geneon a chromosome in a somatic cell or a germ cell is different from itsoriginal structure by deletion of a part of its base sequence, insertionof or replacement with another gene, or the like and cannot produce aprotein capable of functioning as uridine phosphorylase or even if agenetic product is obtained, its protein cannot function as uridinephosphorylase; and specifically, indicates destruction of the functionby gene mutation, such as deletion, insertion or replacement, of thebase sequence of a promoter region or coding region.

The term “non-human animal deficient in a function of a uridinephosphorylase gene on a chromosome”, therefore, means a uridinephosphorylase expression deficient animal created by modifying(manipulating) a uridine phosphorylase gene (mutant uridinephosphorylase gene) in accordance with a genetic engineering procedure.

The “non-human animal” can be any animal having a uridine phosphorylasegene other than human, although a non-human mammal. Examples of thenon-human mammal can include bovines, pigs, sheep, goats, rabbits, dogs,cats, guinea pigs, hamsters, mice, and rats. Among these, rodentsfeaturing relatively short ontogenesis and reproduction cycle and easybreeding, especially mice and rats are preferred from the standpoint ofpreparation of a morbid animal model line.

For the disruption of a uridine phosphorylase gene on a chromosome, itis possible to apply methods which are commonly used for disruptinggenes on chromosomes. Specifically, the function of the uridinephosphorylase gene in an animal and its offspring can be rendereddeficient by cloning the uridine phosphorylase gene to delete thefunction of the gene in vitro and then introducing the deficient geneback into the animal.

Illustrative of methods for introducing a gene into animals are (1)injection of genetic DNA into the embryo of a germ cell in itsprokaryotic stage, (2) transfection of an early embryo with arecombinant retrovirus, and (3) injection of an embryo-derived stem cell(ES cell), which has been caused to undergo homologous recombination,into a blastocyst or into an embryo in its eight cell stage. Thesemethods are all usable for the provision of the non-human animalaccording to the present invention. Nonetheless, the gene introductionmethod making use of an ES cell is preferred in that it is suited fordisrupting a gene by homologous recombination and its permits conductingthe introduction of the ES cell into the gene and the creation of achimeric animal as separate steps.

Therefore, as the non-human animal according to the present invention, achimeric animal can firstly be created by forming a cell having a mutanturidine phosphorylase gene (for example, an ES cell) and transplantingthe cell into an animal; and further, an animal in which the mutanturidine phosphorylase gene is heterogeneously or homogeneously deletedin all the constituent cells thereof can be created by mating thechimeric animal with a different individual.

A description will hereinafter be made in detail about a representativemethod for the production of the non-human animal and its offspringaccording to the present invention.

A mutant of a uridine phosphorylase gene (mutant uridine phosphorylasegene) is obtained by deleting a part of the base sequence of the uridinephosphorylase gene, inserting another gene into the base sequence orreplacing a part of the base sequence with another gene. No particularlimitation is imposed on a position at which the deletion, insertion orreplacement is to be effected, insofar as it is such a position that thedeletion, insertion or replacement makes it impossible to obtain anyfunctioning genetic product. The position can be in any region such as apromoter region, an intron region, an exon region or the like. To assurethe deletion of the expressing ability of the gene, it is preferred tomutate at least a part of the promoter region or the coding region. Itis also possible to insert another gene such as a reporter gene into thedeleted, substituted or inserted position. For example, the uridinephosphorylase gene of a mouse, as illustrated in FIG. 1 and FIG. 2,contains 7 coding exons and a uridine phosphorylase protein is codedover the 7 coding exons. For the deletion of the expressing ability ofthe uridine phosphorylase gene, it is, therefore, possible to delete oneor some of these exons or to insert another gene at any one of thepositions of these exons.

The uridine phosphorylase gene can be either a genome-derived uridinephosphorylase gene isolated or extracted from an animal or cDNA clonedusing a genetic engineering procedure. The cloned uridine phosphorylasegene can be obtained by extracting genomic DNA, for example, from theliver of a mouse, preparing a genomic DNA library from the genomic DNAby a method known per se in the art, and screening the DNA library whileusing as a probe a partial sequence of pre-cloned DNA which codesuridine phosphorylase mRNA, for example, cDNA [in the case of a mouse,Uchida et al., J. Biol. Chem., 270, 12191-12196 (1995)].

Application of an artificial mutation to the above-described uridinephosphorylase gene can be conducted in vitro by usual DNA recombinationtechnology.

When destroying the function of an exon by inserting a reporter gene, itis preferred to insert the reporter gene such that the function of theexon may be expressed under control of a promoter or in a form combinedwith the gene. The term “reporter gene” as used herein indicates a groupof genes, each of which can serve as an index of expression of the gene.In general, structural genes of enzymes which catalyze a photogenicreaction or chromogenic reaction are often used. The reporter gene makesit possible to investigate not only whether the introduced gene has beenexpressed in cells but also which tissue it has been expressed in.Specifically, lacZ (Escherichia coli β-galactosidase gene), cat(chloramphenicol acetyltransferase gene) or the like is used.

Next, a targeting vector is prepared for homologous recombination todelete the function of the uridine phosphorylase gene.

The targeting vector can be designed by deleting the chromosome-deriveduridine phosphorylase gene either in part or in its entirety, insertinganother base sequence into the uridine phosphorylase gene or replacing apart of the uridine phosphorylase gene with another base sequence suchthat the upstream and downstream regions flanking the mutated part havehomologous base sequences as the uridine phosphorylase gene.

The targeting vector can also be designed in such a way that, when areporter gene is inserted in the targeting vector, the base sequence ofthe reporter gene is contained in the non-homologous part of the basesequence of the targeting vector.

Further, the targeting vector may preferably be inserted with markergenes permitting selection of vector-incorporated cells or cells havinghigh possibility of having undergone intended homologous recombination,for examples, a gene widely employed for the selection of drug, such asneomycin-resistant gene (neo), a herpes simplex virus thymidinekinasegene (HSV-tk), or a thymidinekinase gene (tk). For example, the neomycinresistant gene makes it possible to select a target gene by using G418,a neomycin analogue. It is also possible to use, along with a positiveselection marker gene such as the neomycin resistant gene (neo), amarker gene useful for negative selection to selectively remove a targetcell, for example, a thymidinekinase gene (tk) (ganciclovir, FIAU or thelike is used as a selecting agent, and depending on the sensitivity tothe selecting agent, a non-homologous recombinant is selectivelyremoved) or the diphtheria toxin fragment A (DT-A) gene (anon-homologous recombinant is selectively removed by diphtheria toxinexpressed by DT-A).

Preparation of such a targeting vector can be conducted by conventionalDNA recombination technology. For example, a cloned uridinephosphorylase gene is used. This gene is digested with a suitablerestriction enzyme to obtain a fragment, or is partially amplified byPCR to prepare a DNA fragment. The fragment or DNA fragment can then belinked to a linker DNA synthesized by a DNA synthesizer, a fragmentcontaining a reporter gene, a fragment containing a drug-resistancemarker gene and the like in a desired order in accordance with a designas described above.

The homologous recombinational targeting vector prepared as describedabove is next introduced into a suitable cell commonly employed for thecreation of a chimeric animal.

Examples of the cell employed here can include an ovule and anembryo-derived stem cell (ES cell), although an ES cell is preferred inthat is equipped with pluripotency sufficient to differentiate into allkinds of cells in an organism. The introduction of the vector DNA intothe cell can be carried out by a conventional method, for exampleelectroporation, microinjection or calcium phosphate transfection.

An ES cell has been established from an inner cell mass of a mouseblastocyst of 129 cell line, and is a cell line growth and culture ofwhich is feasible while maintaining its undifferentiated state.Concerning mice, a method has been established for introducing a gene byusing an ES cell [Mansour, S. L., et al., Nature, 336, 348 (1988)].Theoretically, culture of an ES cell is considered to be feasible in allspecies of mammals, and research is currently under way to alsoestablish ES cells with respect to J rats, rabbits, and cattle, such aspigs and bovines, in addition to mice. Incidentally, for animal speciesES cells of which are not cultured or are cultured but have not beenestablished to such cell lines as differentiating to germ cells, mutantgenes can be introduced by the above-described method (1) or (2) or alike method.

The mutant uridine phosphorylase gene of the targeting vector can beintroduced into an animal by replacing a uridine phosphorylase gene on achromosome in a cell (for example, an ES cell) of the animal with thebase sequence of the mutant uridine phosphorylase gene of the targetingvector in accordance with homologous gene recombination. At this time,the marker gene and reporter gene inserted in the targeting vector DNAare inserted in the uridine phosphorylase gene in the genome of the EScell.

In the cell with the targeting vector incorporated therein, the markergene, such as a drug resistant gene, in the vector DNA has also beeninserted at the same time so that by culturing the cell for anappropriate period, for example, in the presence of the drug, the cellcan be selected based on the expression of the gene. Among cells soselected, those having undergone mutation by the homologousrecombination can be determined by an analysis making use of Southernhybridization in which the DNA sequence on or adjacent to the uridinephosphorylase gene is used as a probe or by an analysis making use ofPCR in which the DNA sequence on the targeting vector and a DNA sequencein an adjacent region other than the mouse-derived uridine phosphorylasegene employed in the targeting vector are used as primers.

Using the cell with the mutated uridine phosphorylase locus occurredtherein, a chimeric animal is next created in accordance with aprocedure which is generally used for the creation of chimeric animalssuch as injection, cell aggregation or the like.

Described specifically, a chimeric animal—which is composed of cellshaving the normal uridine phosphorylase focuses and cells having themutated uridine phosphorylase locuses—can be obtained by injecting acell (for example, an ES cell) with the mutated locus occurred thereininto a non-human animal embryo or blastocyst in an appropriate earlystage of embryo formation, for example, in its 8-cell stage andtransplanting the resulting embryo in the uterus of a pseudocyeticanimal. Selection as to what line of animal a host embryo is to beobtained from should be made such that cells to be derived from the EScell and those to be derived from the host embryo would be successfullydistinguished depending on a phenotype such as fur colors in accordancewith a usual method.

When some of germ cells of the chimeric animal have the mutant uridinephosphorylase locuses, individuals the tissues of which are all composedof cells having the mutant uridine phosphorylase locuses (uridinephosphorylase expression deficient animals) can be selected by adiscrimination method, which relies upon the fur color or the like, froma group of individuals obtained by mating a chimeric individual with anormal individual.

When a chimeric mouse obtained using, for example, a mouse ES cell ofthe J1 strain and a host embryo of the C57BL/J line is mated with aC57BL/6J-line mouse, offspring to be ejected will present the same wildcolor (agouti) as the mouse from which the ES cell was derived if germcells of the chimeric mouse are derived from the recombinant ES cell,but present the same black color as the mouse from which the host embryowas derived if the germ cells of the chimeric mouse ware derived fromthe host embryo. The deleted expression of the uridine phosphorylasegene can be confirmed by performing Southern blot or PCR analysis oftail DNA from new born and fed mice.

Further, mating between F1 heterozygous expression deficient offspringthemselves makes it possible to obtain both F2 heterozygous expressiondeficient offspring and F2 homozygous expression deficient offspring.Non-human animals according to the present invention can embracechimeric animals, F1 heterozygous expression deficient animals and F2homozylous expression deficient animals. F2 homozylous expressiondeficient animals are, however, preferred from the standpoint of effectsof the expression deficiency of the uridine phosphorylase gene.

Transformed non-human animals obtained as described above are deficientin the ability to express uridine phosphorylase genes on chromosomes insomatic cells and germ cells, and the deficiency is genetically stable.

EXAMPLES

The present invention will hereinafter be described specifically basedon Examples.

Example 1

Preparation of Targeting Vector of the Mouse Uridine Phosphorylase GeneDNA

A recombinant phage library of lambda FIXII with genomic DNA of a 129SVmouse liver liked thereto (product of Stratagene LLC) was obtained andtransfected to Escherichia coli LE392. With respect to the resultingmouse genomic library, hybridization was conducted using cDNA of themouse uridine phosphorylase gene as a probe [Uchida et al., J. Biol.Chem., 270, 12191-12196 (1995)]. As a result of screening on 1×10⁶plaques, 2 positive clones were obtained (FIG. 1: clone No. 7 and cloneNo. 6). Using these, an approx. 1.8 kb SacI-SacI fragment containingintron 1 of the uridine phosphorylase gene (FIG. 1: DNA 1) and anapprox. 7.0 kb XbaI-XbaI fragment containing exons 3-7 (FIG. 1: DNA 2)were subcloned.

Next, preparation of a targeting vector for disrupting the structure ofthe uridine phosphorylase gene was conducted. A neomycin resistant gene(pGK-neo) was inserted into pBluescrptIISK(+), and the diphtheria toxinfragment A gene (DT-A) was inserted in a direction opposite to thenormal direction of its transcription on the upstream 5′-end side.Further, the approx. 1.8 kb SacI-SacI fragment with intron 1 of theuridine phosphorylase gene contained therein (FIG. 1: DNA 1) wasinserted as a segment homologous to the genomic DNA between pGK-neo andDT-A, and the approx. 7.0 kb XbaI-XbaI fragment with coding exons 3-7contained therein (FIGS. 1 and 2: DNA 2) was inserted on the downstream3′ side of pGK-neo. As a result, UP-neo (FIGS. 1 and 2) was obtained.Furthermore, an SA-NLacZ gene with a splicing acceptor (SA) and anuclear transport signal added thereto was inserted as anexpression-analyzing reporter gene between DNA1 and pGK-neo to obtainUP-LacZ (FIGS. 1 and 2). Upon introduction into ES cells, the constructswere both digested and linearized with a restriction enzyme NotI toobtain targeting vectors.

Example 2

Deletion of the Uridine Phosphorylase Gene from ES Cells by theIntroduction of DNA for Homologous Recombination

The two kinds of DNAs for homologous recombination (UP-neo and UP-LacZ;25 μg, each) were separately suspended in aliquots of an electroporatingbuffer, each of said aliquots containing 2×10⁷ mouse ES cells (J1strain), and under conditions of 400 V/cm field strength and 25 μFcapacitance, gene introduction was conducted. Selective incubation wasconducted at a G418 (“Genetisin”, product of GIBCO BRL) concentration of175 μg/mL from the 46^(th) hour after the introduction of UP-neo andfrom the 44^(th) hour after the introduction of UP-LacZ, respectively.

Each G418 colony was transferred to a 96-well microplate, each well ofwhich contained 0.05% trypsin-HBS-EDTA solution (40 μL), by a micropipetfrom the 184^(th) hour after the introduction of the corresponding gene.After the colony was treated for 5 minutes, it was pipetted to providesingle cells. Those single cells were halved, and were then transferredto 96-well microplates, followed by incubation. After sufficientproliferation, they were transferred to 24-well microplates andincubated further. A half of the cells was stored in liquefied nitrogen,while the remaining half of the cells used for extracting DNA to be usedfor the detection of a recombinant by Southern hybridization.

The DNA extraction was conducted by a method known per se in the art. Asthe samples were many, DNA precipitated by ProteinaseK treatment andisopropanol treatment was digested by EcoRI, followed by Southernhybridization. As a probe, an approx. 1.3 kb EcoRI-SacI fragmentsituated further upstream of the 5′-side homologous region employed asthe targeting vector was used as illustrated in FIG. 1. When detected bySouthern hybridization, use of that probe was expected to result in theappearance of a band at 10 kb in the case of the non-homologousrecombinant ES cells, at 5.1 kb in the case of the ES cells undergonehomologous recombination by UP-neo because of the introduction ofEcoRI-digested sites upon insertion of the neomycin resistant gene, orat 6.1 kb in the case of UP-LacZ.

The results of the hybridization were analyzed by Bio-Image AnalyzerBAS2000 (Fuji). As the frequency of homologous recombination, 1 out of70 clones underwent homologous recombination in the case of UP-neo,while 4 out of 135 clones underwent homologous recombination in the caseof UP-LacZ (FIG. 3). A cell, bands of a mutant locus and a wild locus inwhich appeared at substantially the same intensity, was selected andintroduced in a blastocyst.

Example 3

Creation of Chimeric Mice and Selection of Uridine PhosphorylaseExpression Deficient Mouse

A chimeric mouse was prepared by the method reported by Bradley et al.[Bradley, A., Production and analysis of chimeric mice interatocarcinomas and embryonic stem cells; A practical approach. E. J.Roberson ed. (Oxford: IRL press), 113-151(1987)] or by the methodproposed by Gossler et al. [Gossler, A., Doetschman, T., Korn, R.,Serfling, E., and Kemler, R., Transgenesis by means ofblastocyst-derived embryonic stem cell lines, Proc. Natl. Acad. Sci.USA, 83, 9065-9069 (1986)]. On the 3.5^(th) day after copulation withC57BL/6, a blastocyst was obtained from the oviduct, and in its cavity,about 7 homologous recombinant ES cells were injected. The blastocystwith the ES cells injected therein was returned to the uterus of apseudopregnated ICR mouse. From chimeric mice born from the fostermother, two mice with a fur color lightened from the inherent blackcolor, said fur color lightening being presumably attributable to alarge contribution by the ES cell, were selected, and those selectedmice were allowed to mate with C57BL/6. For an agouti colored one ofnewly born mice, Southern blot analysis of tailed DNA was performed bythe general method. After treated with proteinase K, the DNA in the tailtissue was extracted with chloroform, precipitated in ethanol, and thendissolved in TE solution. Mice having locuses, which had undergonerecombination, were selected, and were allowed to mate with each otherto obtain mice having homozygous recombinant uridine phosphorylaselocuses.

Using the 1.3 kb EcoRI-SacI fragment as a probe, Southern hybridizationwas then conducted. Bands appeared at 6.1 kb and 10 kb, respectively, sothat uridine phosphorylase on one of the chromosomes was determined tohave been destroyed. Twenty-one (21) mice of the agouti color wereobtained concerning UP-neo, while 36 mice of the agouti color wereobtained with respect to UP-LacZ. When analyzed by Southernhybridization, 11 UP-neo mutant mice showed bands at 5.1 kb and 10 kb,respectively, and 16 UP-LacZ mutant mice presented bands at 6.1 kb and10 kb, respectively. Those mice were then allowed to mutually mate.Eventually, UP-neo mutant mice showing a band only at 5.1 kb and UP-LacZmutant mice presenting a band only at 6.1 kb, that is, mice havinghomozygous mutant uridine phosphorylase locuses were obtained (FIG. 4).Mice so born did not show any appreciable abnormality. It has thereforebeen determined that such mice are born absolutely healthy. As micehaving gene types of UP +/+, +/− and −/− were born at a ratio of approx.1:2:1 between the litter mates, it has also been determined that UP −/−mice are also born absolutely healthy irrespective of the geneticbackground.

Example 4

Uridine Phosphorylase Activity in Normal and Mutant Mice

Mice having homozygous mutant uridine phosphorylase genes wereinvestigated for the existence or non-existence of uridine phosphorylaseactivity. Briefly, small intestine and liver tissues from uridinephosphorylase deficient mice and parental mice (normal) as a control,showing full expression of uridine phosphorylase gene, were isolated andhomogenized with 4 volumes of a buffer (50 mM Tris-HCl, 10 mM2-mercaptoethanol, 25 mM KCl, 5 mM MgCl₂). Subsequent to centrifugationunder 105,000 g for 60 minutes, the activity was measured using itssupernatant. Uridine phosphorylase activity was measured based on[³H]uridine formed from [³H]uracil by the method reported by Ikenaka etal. [Ikenaka et al., Metabolism of pyrimidine nucleotides in varioustissues and tumor cells from rodents. Gann. 72(4), 590-7 (August,1981)]. At the same time, the activity of thymidine phosphorylase, theother enzyme in the pyrimidine nucleoside phosphorylase group, was alsomeasured based on [³H]thymine formed from [³H]thymidine. The results arepresented-in Table 1.

TABLE 1 Uridine phosphorylase Thymidine phosphorylase activity¹⁾activity²⁾ (nmol/min/mg protein) (nmol/min/mg protein) Small Small Micen intestine Liver intestine Liver Non-recombinant (normal) mice 15 20.99± 8.18 0.27 ± 0.12 2.31 ± 0.94 1.11 ± 0.06 Uridine phosphorylase 15 0.44 ± 0.19 0.04 ± 0.05 0.68 ± 0.40 1.01 ± 0.58 expression deficientmice ¹⁾Measured based on [³H] uracil formed from [³R] uridine.²⁾Measured based on [³H] thymine formed from [³H] thymidine.

As a result, the uridine phosphorylase activity in the small intestinetissue was high in the non-recombinant (normal) mice but was markedlyreduced and lost in the uridine phosphorylase expression deficient mice.In the liver tissues of the normal mice, uridine phosphorylase activitywas low but thymidine phosphorylase was expressed high. In the uridinephosphorylase expression deficient mice, on the other hand, uridinephosphorylase activity was substantially lost in the liver tissues.

Example 5

Expression of Uridine Phosphorylase Protein in the Small Intestines ofNormal and Uridine Phosphorylase Expression Deficient Mice

From 5 normal mice and 5 uridine phosphorylase expression deficientmice, small intestine tissues were enucleated, respectively. From eachsmall intestine tissue, a protein extract was prepared in accordancewith the above-described procedure. The protein extract was adjusted toa protein content of 5 mg/mL, and was investigated for the expression ofuridine phosphorylase protein by Western blotting. For the detection,anti-mouse uridine phosphorylase polyclonal antibody was used. As isshown in FIG. 5, practically no band of uridine phosphorylase wasdetected in the uridine phosphorylase expression deficient mice.

Example 6

Blood Uridine Levels in the Intestines of Normal and UridinePhosphorylase Expression Deficient Mice

Blood samples were collected from normal and uridine phosphorylaseexpression deficient mice, respectively. From each blood sample, serumwas separated, followed by the addition of 9 volumes of ice-coldmethanol. The resulting mixture was left over in ice for 30 minutes tohave proteins fully precipitated. The mixture was then centrifuged and,after the supernatant so obtained was caused to evaporate to dryness at60° C. under a nitrogen gas stream, purified water (200 μL) was added tothe residue. The thus-prepared mixture was filtered through a 0.45-μmfilter. The concentrations of uridine, thymidine and cytidine weredetermined by HPLC. The results are presented in Table 2.

TABLE 2 Uridine Thymidine Cytidine concentration concentrationconcentration Mice (nmol/mL ± SD) (nmol/mL ± SD) (nmol/mL ± SD) Non- 5.33 ± 1.34 0.78 ± 0.05 6.00 ± 1.06 recombinant (normal) mice Uridine27.26 ± 2.78 1.35 ± 0.01 8.94 ± 0.69 phosphorylase expression deficientmice

As is shown in Table 2, the serum uridine level was 5.33 nmol/mL in thenormal mice and 27.26 nmol/mL in the uridine phosphorylase expressiondeficient mice. An increase as much as about 5 times was thereforeobserved in the uridine phosphorylase expression deficient mice.Concerning the concentrations of thymidine and cytidine, the other bloodnucleosides, only slight increases were observed in the uridinephosphorylase expression deficient mice than in the normal mice, and nosubstantial changes were confirmed by the knockout of the uridinephosphorylase expressing function.

Example 7

Metabolic Kinetics of Nucleoside Anticancer Agents in Normal and UridinePhosphorylase Expression Deficient Mice

Changes in the metabolic kinetics of nucleoside anticancer agents by theknockout of the uridine phosphorylase expressing function wereinvestigated using 5-fluorouridine (FUR) and 5-trifluorothymidine(F₃dThd).

Normal and uridine phosphorylase expression deficient mice (n=3 pergroup) were orally administered with 50 mg/kg of FUR or 10 mg/kg ofF₃dThd, and blood samples were collected with time. After serum wasseparated from each blood sample, the concentration of FUR or F₃dThd inthe serum was determined by HPLC.

The results are presented in Table 3.

TABLE 3 Time FUR concentration F₃dThd concentration Mice (min) (nmol/mL± SD) (nmol/mL ± SD) Non-recombinant (normal) mice 15 1.22 ± 0.12 16.61± 5.76  30 1.26 ± 0.51 8.19 ± 4.29 60 2.08 ± 0.98 2.64 ± 1.17 120 Nottested 0.10 ± 0.08 Uridine phosphorylase expression 15 5.95 ± 1.15 16.75± 2.94  deficient mice 30 5.05 ± 2.48 13.42 ± 2.17  60 3.33 ± 1.30 6.52± 5.24 120 Not tested 1.78 ± 0.94 Each value is an average ± SD of n =3.

As is shown in Table 3, when FUR was orally administered, theconcentration of FUR in the uridine phosphorylase expression deficientmice was about 5 times as high as that in the normal mice (the values at15 to 30 minutes after the administration). It has, therefore, beensubstantiated that as a result of the deletion of uridine phosphorylaseactivity, the decomposition of FUR in the body is inhibited. In the caseof F₃dThd, on the other hand, the concentration of F₃dThd in the uridinephosphorylase expression deficient mice was also 1.6 to 17.8 times ashigh as the corresponding value in the normal mice from 30 minutes to120 minutes after its oral administration. It was, hence, ascertainedthat the proportion of F₃dThd decomposed by uridine phosphorylase wasreduced.

From the above results, mice deficient in the function of uridinephosphorylase genes are considered to be human-resembling models, and inconnection with pyrimidine nucleoside anticancer agents, their metabolickinetics in human are predictable from their metabolic kinetics in thesemice.

INDUSTRIAL APPLICABILITY

By the present invention, genetically stable, uridine phosphorylaseexpression deficient animals can be obtained. These animals make itpossible to elucidate pathologic functions or activities such as nucleicacid dysbolism and also to predict utility of pyrimidine nucleosideantimetabolites in human, and are useful as experimental animals.

What is claimed is:
 1. A mouse comprising in its genome homozygousinactivated uridine phosphorylase gene locuses, or an offspring thereof,wherein said mouse is deficient in the expression of uridinephosphorylase and exhibits a decreased metabolism of a nucleotideanticancer agent or an antimetabolite, or both.
 2. The mouse of claim 1or an offspring thereof, wherein the function of said uridinephosphorylase gene has been rendered deficient by deleting a part of aDNA sequence of said uridine phosphorylase gene or by inserting orreplacing another gene into or at a site of said DNA sequence.
 3. Themouse of claim 2 or an offspring thereof, wherein said another gene is amarker gene or a reporter gene.
 4. The mouse of claim 3, or an offspringthereof, comprising a marker gene that is a neomycin resistance gene. 5.The mouse of claim 1 or an offspring thereof comprising a mouse uridinephosphorylase gene with a neomycin resistance gene inserted in place ofa second coding exon in said mouse uridine phosphorylase gene.
 6. Amethod for measuring the kinetics of an anticancer agent or anantimetabolite comprising administering said anticancer agent orantimetabolite to the mouse of claim 1 and measuring the metabolickinetics of said anticancer agent or antimetabolite.
 7. The method ofclaim 6, wherein said anticancer agent or antimetabolite is a pyrimidinenucleoside.
 8. A method for making a mouse comprising in its genomehomozygous inactivated uridine phosphorylase gene locuses, wherein saidmouse is deficient in the expression of uridine phosphorylase andexhibits a decreased metabolism of a nucleotide anticancer agent or anantimetabolite, or both, comprising: mating two heterozygous mice eachcomprising an inactivated uridine phosphorylase gene, selecting anoffspring comprising in its genome homozygous inactivated uridinephosphorylase gene locuses, which is deficient in the expression ofuridine phosphorylase and which exhibits a decreased metabolism of anucleotide anticancer agent or an antimetabolite, or both.